Cartilage proteoglycan-induced arthritis in BALB/c mice. Antibodies that recognize human and mouse cartilage proteoglycan and can cause depletion of cartilage proteoglycan with little or no synovitis.

Human fetal cartilage proteoglycan (PG) induces the development of an erosive polyarthritis and spondylitis in BALB/c mice. We have examined the properties of 3 monoclonal antibodies (MAb) to human fetal cartilage PG isolated from immunized mice that cross-react with mouse cartilage PG. Compared with sera from arthritic mice, which contain antibodies reactive with keratan sulfate, MAb 202 (IgG1) reacted only with a protein-related epitope that is distributed on both hyaluronic acid-binding and chondroitin sulfate-attachment regions. MAb 813 (IgG1) reacted with the same fragments and recognized an epitope with the immunologic characteristics of keratan sulfate. MAb 945 (IgM) remains to be further characterized. Introduction of hybridomas secreting MAb 202 and MAb 945 into irradiated mice resulted in the loss of PG from articular cartilage and from growth plate cartilage (with MAb 202 only), as revealed by a loss of staining with toluidine blue. There was no synovial hyperplasia with MAb 202, but some hyperplasia and mononuclear cell infiltration was seen with MAb 945. This was accompanied by the binding of immunoglobulins to articular cartilage, as demonstrated by immunofluorescence. The hybridoma secreting MAb 813 produced no cartilage changes or synovitis, and there was no immunoglobulin binding to cartilage. Polymorphonuclear leukocyte infiltration was never observed with these antibodies. These studies indicate that MAb reactive with mouse cartilage PG can cause the depletion of PG from hyaline cartilage by mechanisms that may be both complement dependent and complement independent. Antibodies may serve to release and expose PG antigen to immune cells, as well as causing a loss of the mechanical properties of cartilage that are PG dependent.

Iliac crest growth plate analysis in slipped capital femoral epiphysis.

Previous studies have demonstrated numerous histologic and histochemical abnormalities in the involved growth plate of patients with slipped capital femoral epiphysis (SCFE). Whether these changes are primary or secondary with regard to the slip is unknown. We examined an uninvolved site of patients with SCFE and controls, the iliac crest growth plate, by histochemical means for general morphology, proteoglycans, glycoproteins, and collagen fibril orientation. No differences were noted between the two groups studied. This observation suggests that previously reported slipped capital femoral epiphyseal abnormalities may be a result of, rather than a cause of, the slip in this condition.

Use of histochemical techniques in the characterisation of osteochondroses affecting pigs.

Growth cartilages with dyschondroplastic foci (osteochondrosis) or areas of chondrolysis were selected from the ribs and bones of the appendicular skeleton of 132 commercial pigs euthanased between one and 169 days old or at a liveweight of 100 kg. Histochemical staining techniques that identified proteoglycans, collagen and deposits of calcium enhanced the visualisation of the lesions, were valuable for recognising the distribution of lesions, and helped to elucidate the development of the lesions. On the basis of the histochemical and morphological differences, it was considered that the lesions associated with growth plates and the lesions associated with articular-epiphyseal cartilage complexes should be considered as different entities. Lesions were identified in the articular-epiphyseal cartilage complexes of pigs at 15 days old, earlier than has been reported previously. Toluidine blue and safranin O were more useful than alcian blue and, in terms of staining intensity, toluidine blue gave more consistent results than safranin O.

Determinación de la edad en que cierran las placas de crecimiento en las rodillas de conejos de raza Nueva Zelanda / The age of physeal closure in the knee of New Zelanda rabbits

La finalidad de este estudio fue determinar la edad del cierre de las placas de crecimiento en la zona de la rodilla de los conejos de raza Nueva Zelanda. Se estudiaron 124 conejos de diferentes edades mediante radiografías simples y cortes histológicos. Se concluyó que el cierre fisiario distal del fémur acontece entre 131 y 166 días. Para la placa de crecimiento proximal de la tibia, el cierre fisiario tiene lugar entre 176 y 197 días. Los estudios radiológicos e histológicos discreparon para el fémur en 3.6% de los casos, para la tibia el 10.9%, y para el peroné el 16.4%; es decir, casos en los que la fisis no era visible radiológica pero si histológicamente.

Visualization of sulfur-containing components associated with proliferating chondrocytes from rat epiphyseal growth plate cartilage: possible proteoglycan and collagen co-migration.

Electron microscopy of epiphyseal growth plate cartilage from normal 4-5-week-old rats has revealed extensive fibrillar aggregates and globules in the pericellular spaces of proliferating chondrocytes. These cells contained small globules and diffusely coiled, fine filaments located within large, membrane-invested vacuoles. All such structures were observed after a variety of different tissue fixation regimes, including glutaraldehyde, osmium tetroxide, and potassium pyroantimonate. The fibrillar aggregates and globules were often overlapping and intermeshed and extended to 0.5 micron in length from their point of origin at cell membranes. Vacuoles were usually found at the periphery of cells, and some, by membrane fusion with the cell envelope, appeared contiguous with extracellular spaces wherein their contents could be discharged. Fine filaments and globules were occasionally observed in the Golgi complex and cisternae of endoplasmic reticulum of the chondrocytes. Further characterization of the cellular and pericellular components by electron microscopic radioautography, electron probe microanalysis, and electron spectroscopic imaging indicated the presence of sulfur, a result suggesting these aggregates, filaments, and globules in part represent proteoglycans in various stages of synthesis, secretion, and assembly. Additional radioautography utilizing 3H-proline implied that filament bundles are also composed of collagen, a result posing the possibility that this protein and the putative proteoglycans may co-migrate both intracellularly and within pericellular matrices. In extracellular matrices adjacent to cell lacunae, the fibrillar aggregates appeared in close association with typical collagen type II fibrils, an observation providing evidence for proteoglycan-collagen network formation in this region of the rat epiphysis. These microscopic and analytical data in situ would support certain studies in vitro of proteoglycan-collagen type II and IX association and are important in describing the interaction of such cartilage components ultimately involved in matrix formation.

Isolation of bovine type X collagen and immunolocalization in growth-plate cartilage.

Type X collagen was extracted with 1 M-NaCl and 10 mM-dithiothreitol at neutral pH from fetal-bovine growth cartilage and purified to homogeneity by using f.p.l.c. gel filtration on a Superose 12 column, followed by ion-exchange chromatography on a Mono Q column. The purified protein migrates in SDS/polyacrylamide gels with an apparent Mr of 58,000 under reducing conditions and as a high-Mr oligomer in its unreduced form. The amino acid composition is similar to the published composition of chick type X collagen. Pepsin digestion at 4 degrees C decreases the Mr of the monomer to 43,000; purified bacterial collagenase digests most of the molecule, leaving a non-collagenous domain of apparent Mr 15,000, which probably represents the C-terminal globular domain. The IgG fraction from a rabbit antiserum raised against purified bovine type X collagen was specific for this collagen by the criteria of e.l.i.s.a. and immunoblotting after immunoabsorption with collagen types I, II, IX and XI. Immunofluorescence localization of type X collagen in sections of fetal-bovine and human cartilage was possible after acetone fixation of sections and hyaluronidase treatment. Type X collagen was restricted to the zone of hypertrophic and calcified cartilage inside the bone spicules of the growth plate.

An immunohistochemical study of localization of type I and type II collagens in mandibular condylar cartilage compared with tibial growth plate.

Immunohistochemical localization of type I and type II collagens was examined in the rat mandibular condylar cartilage (as the secondary cartilage) and compared with that in the tibial growth plate (as the primary cartilage) using plastic embedded tissues. In the condylar cartilage, type I collagen was present not only in the extracellular matrix (ECM) of the fibrous, proliferative, and transitional cell layers, but also in the ECM of the maturative and hypertrophic cell layers. Type II collagen was present in the ECM of the maturative and hypertrophic cell layers. In the growth plate, type II collagen was present in the ECM of whole cartilaginous layers; type I collagen was not present in the cartilage but in the perichondrium and the bone matrices. These results indicate that differences exist in the components of the ECM between the primary and secondary cartilages. It is suggested that these two tissues differ in the developmental processes and/or in the reactions to their own local functional needs.

Histological, biochemical, and MRI studies of the growth plate in congenital coxa vara.

Magnetic resonance imaging (MRI) studies in two patients with congenital coxa vara revealed a widened growth plate with expansion of cartilage mediodistally between the capital femoral epiphysis and metaphysis. They did not reveal slippage. Histologic studies showed that the growth plate appeared to be composed merely of irregularly and scarcely distributed germinal cartilage cells with abundant matrix. The histologic studies resembled the histopathologic reports of metaphyseal chondrodysplasia Schmid type, but only with regard to the proximal femoral growth plate. Determination of the sulfur concentration of the cartilaginous matrix was within normal range.

Effect of swimming on bone growth and development in young rats.

The effect of chronic swimming on bone modelling was studied. Forty female Sabra rats (5 weeks old) were randomly assigned to the following experimental groups: 30 rats were trained to swim (water bath 35 +/- 1 degree C, one h daily, five times a week) for 20 weeks--20 of them loaded with lead weights (1% body weight) while the rest (10 animals) swam load free. Ten sedentary rats matched for age and weight served as controls. At the end of the twenty-week swimming period, all rats were sacrificed, both humeri bones were dissected and prepared for the following examinations: morphometric, bone density (BD), bone mineral content (BMC), compression tests and cross-sectional geometrical parameters, histomorphometry and biochemical analysis of minerals (Ca, Pi, Mg, Zn). All measured parameters were found to be significantly higher (P less than 0.05) in the swimming rats irrespective of load, as compared with the controls. Bone weight was higher by 19%, bone volume by 11%, bone length by 2.8%, cortical area by 16%, BD by 7% and BMC by 15%. The compression breaking force at the distal shaft of the humerus was higher by 24% in the trained group, while the ultimate compressive stress was not significantly different. Maximal and minimal moment of inertia at the distal diaphysis were 33.4 and 40% higher, respectively, for the swimming groups than the controls. Ca, Pi, Mg and Zn levels per total humeral bone were significantly higher in the exercising rats. The histomorphometry and cross-sectional data emphasize longitudinal and transversal growth. These data indicate that swimming exercise exerts a positive effect on bone growth and development in young rats.

Immunoreactive calbindin-D9K localization in matrix vesicle-initiated calcification in rat epiphyseal cartilage: an immunoelectron microscope study.

Calbindin-D9K immunoreactivity was localized by electron microscopy in rat calcifying epiphyseal plate cartilage. Antigen-antibody reaction sites were visualized by the presence of protein A-gold complex particles on undecalcified material embedded in Lowicryl K4M. Immunoreactive calbindin-D9K was found in the hyaloplasm of hypertrophic chondrocytes and inside and at the ends of their cell processes. It was localized outside the cells, inside matrix vesicles (MVs), often against the inner face of the delimiting membrane, and inside the trilaminar membrane. Immunoreactive calbindin-D9K appeared to be extruded from the chondrocytes into the matrix vesicles when the latter were formed during the budding of cell processes. In calcifying MVs, gold particles were detected over the needle-shaped crystallites and often over the crystallites lying against the inner leaflet of the vesicular membrane. At a later stage of matrix vesicle calcification after MV membrane disruption, the number of gold particles remained unchanged over the clusters of crystallites at the loci from which the crystallites appeared to have grown and radiated. At a yet more advanced stage of calcification, they remained in the same areas, which were limited to the lateral edges of calcified cartilage longitudinal septa. These results suggest that immunoreactive calbindin-D9K plays a role in calcium input to matrix vesicles and may be involved in matrix vesicle calcification, perhaps in the initial event of matrix vesicle crystal nucleation.

Ultrastructural demonstration of increased sulfated proteoglycans and calcium associated with chondrocyte cytoplasmic processes and matrix vesicles in rat growth plate cartilage.

A monoclonal antibody (3-B-3) to chondroitin 6-sulfated proteoglycan was used with immunoperoxidase electron microscopy to study the relationship of chondrocyte cytoplasmic processes and matrix vesicles in rat epiphyseal growth plate cartilage. Immunoperoxidase staining of the chondrocyte plasmalemma was found at all levels in the growth plate and was most prominent in the hypertrophic zone. The plasmalemma and matrix of the cytoplasmic process often demonstrated stronger reactivity than the remainder of the cell surface. Matrix vesicles showed weak to strong surface or internal reactivity. The majority of them stained very similarly to the cytoplasmic process. X-ray microanalysis of specimens processed by rapid freezing and freeze substitution confirmed that both sulfur and calcium were localized within or in close association with both the cytoplasmic process and the matrix vesicle, suggesting a chemical combination of calcium with sulfated proteoglycans at both sites. These results indicate that there is a selective increase in the concentration of membrane-associated sulfated proteoglycan and calcium in the cell process, from which matrix vesicles may be released into the extracellular matrix.

Demonstration of growth hormone receptors in cultured rat epiphyseal chondrocytes by specific binding of growth hormone and immunohistochemistry.

Growth hormone has been reported to exert direct effects on rat and rabbit epiphyseal chondrocytes in vitro, indicating that GH interacts with specific receptors on these cells. To investigate this possibility, binding of GH to cultured rat epiphyseal chondrocytes was studied under various experimental conditions. Chondrocytes were isolated enzymatically from epiphyseal growth plates of the proximal tibia of 20-day-old male rats and were cultured in monolayer in Ham's F-12 medium supplemented with 10% calf serum and 1% of a serum substitute. The cells were seeded at various densities (25,000-200,000 cells/well) and cultured for 5-16 days. Twenty-four hours before binding experiments, the medium containing calf serum was changed for one containing serum obtained from hypophysectomized rats, in order to avoid binding of GH present in the calf serum. Binding was studied by incubating 125I-labelled human GH (hGH) with the cells in the presence or absence of various concentrations of unlabelled hGH, bovine GH (bGH), rat GH (rGH) and ovine prolactin (oPRL). Specific binding could be demonstrated in cells cultured for 5-16 days. Binding was dependent upon time and temperature, and maximal binding was obtained by incubating the labelled hormone for 4-6 h at 24 degrees C. An increase in binding was noted between 7 and 12 days in culture. In cells cultured for 12 days, addition of unlabelled hGH, bGH or rGH caused a dose-dependent displacement of 125I-labelled hGH, whereas oPRL was ineffective. Scatchard analysis resulted in a linear plot, and the number of binding sites/cell was approximately 5700, with a dissociation constant of 0.46 nmol/l.(ABSTRACT TRUNCATED AT 250 WORDS)

Freeze-substitution staining of rat growth plate cartilage with alcian blue for electron microscopic study of proteoglycans.

To selectively stain polyanionic macromolecules of growth plate cartilage and to prevent artifacts induced by aqueous fixation, proximal tibial growth plates were excised from rats, slam-frozen, and freeze-substituted in 100% methanol containing the cationic dye Alcian blue. Electron microscopic examination showed the tissue stained with Alcian blue to be comparable in ultrastructural preservation to tissues slam-frozen and freeze-substituted in the absence of Alcian blue. The extracellular matrix exhibited a characteristic staining pattern when stained by this method. The pericellular rim was identified as a band of varying width encircling the chondrocyte and its cell processes. Peripheral to the pericellular rim the heterogeneity of staining within the extracellular matrix increased, taking the form of polymorphic densities. X-ray microanalysis showed that the visual interpretation of electron density was related to the concentration of copper present, and that the concentration of sulfur was variable in the pericellular rim and in the interterritorial matrix. The difficulties associated with aqueous fixation and staining procedures are discussed in contrast to the improved preservation achieved by cryogenic methods.

Immunohistochemical localization of type II collagen in cartilage-forming tumors.

The presence and distribution of type II collagen was studied in 36 cartilage and cartilage-related tumors, including five osteosarcomas and one chordoma. A monoclonal antibody prepared from chicken type II collagen was used with paraffin sections, employing the ABC (avidin biotinylated horseradish peroxidase complex) peroxidase technique. Fetal cartilage and fracture callus were used as control materials. Type II collagen was present in the matrix of all the cartilage tumors. The reaction was strongest in areas of well-differentiated cartilage and weakest in the poorly differentiated tissue of high-grade chondrosarcomas. Areas of mineralization or ossification, and areas of eosinophilic, fibrous, or degenerated cartilage gave a negative reaction.

Problems in the immunolocalization of type IX collagen in fetal calf cartilage using a monoclonal antibody.

Monoclonal antibodies were prepared against the pepsin-resistant fragments (X1-X3) of bovine type IX collagen. One of the five hybridomas that gave a positive reaction in an enzyme-linked immunosorbent assay was selected (H1a) for structural analysis and immunolocalization of type IX collagen. The location of the epitope for H1a was deducted from immunoblots and electron microscopic observations after rotary shadowing. The H1a antibody binds to one end of the longest X2, X3, X4 molecules, and preferentially 40-55nm from one end of X1 molecules thus, on or near the noncollagenous domain, NC2. Different immunolocalizations of type IX collagen in the superficial, middle and deep zones of fetal calf epiphyseal cartilage were observed depending on the thickness of the section and on hyaluronidase digestion conditions. In the middle and deep zones, staining with H1a throughout the matrix was obtained only with thin sections (5 microns) and digestion for 1 h at 37 degrees C. With thick sections (15 microns) or with digestion for 1 h at 24 degrees C, staining was restricted to the pericellular regions. Staining throughout the matrix was obtained in the superficial zone under all experimental conditions. Without hyaluronidase treatment, no immunofluorescent staining was seen with either H1a or polyclonal antibody to type II collagen, indicating that type IX collagen is present throughout the matrix in the different zones of fetal calf cartilage. This result is in good accordance with the recent demonstration of common cross-links between type II and type IX collagen in chicken and bovine cartilage. However, the preferential unmasking of type IX collagen antigenic sites in the pericellular regions of middle and deep zones of fetal calf cartilage does not preclude the presence in that region of a special pericellular organization of the collagenous network.

Covalent interactions of type IX collagen in cartilage.

The cross-linking of type IX collagen in fetal bovine cartilage was investigated. The main cross-link was dihydroxy-lysinonorleucine (borohydride-reduced) with a lesser amount of the mature cross-link, pyridinoline. Dihydroxylysinonorleucine was present in all three chains of the COL2 domain of the type IX molecule, but only two of them contained pyridinoline even in mature cartilage. Amino acid sequence analysis of individual tryptic peptides that contained 3H-labeled cross-links showed that they had derived from sites of covalent interaction between type IX collagen and the telopeptide sequences of type II collagen. One two-chained peptide was a helical sequence of alpha 2 (IX) COL2 linked to an alpha 1 (II) N-telopeptide. A second peptide was a different helical sequence from another type IX chain linked to an alpha 1(II) c-telopeptide. This latter helical sequence was also the principal site of pyridinoline cross-linking in type IX collagen.

Type X collagen in avian tibial dyschondroplasia.

Tibial dyschondroplasia (TD) is an abnormality of the growth plate of growing chicks characterized by the presence of a mass of nonvascularized, nonmineralized cartilage. To determine if the morphologic changes observed in TD lesions are accompanied by biochemical abnormalities of the cartilage matrix, we performed quantitative and qualitative analyses of collagen in affected cartilage, placing special emphasis on the study of type X collagen, a recently described molecular species that may play a role in endochondral ossification. Collagen from TD lesions of 25-day-old chicks and from growth plate cartilage of normal age-matched chickens and from hypertrophic cones of 1-day-old chicks was sequentially extracted, purified and characterized. No differences were noted in total collagen content and extractibility between these tissues. Determination of the relative amounts of the various collagens extracted showed that type X collagen in the TD lesion was markedly reduced since it was less than half of that found in pure hypertrophic cartilage. These results are consistent with previous morphologic and biochemical studies which suggest a failure of the chondrocytes to fully achieve maturation and hypertrophy. No differences were demonstrated in the CNBr peptide patterns or amino acid composition of type X collagen from normal or TD and hypertrophic cartilages, suggesting that the low content of type X collagen in TD lesion was due to alterations in type X collagen metabolism rather than to structural abnormalities in these molecules.

Histochemical, immunofluorescence, and ultrastructural differences in fetal cartilage among three genetically distinct chondrodystrophic mice.

The severe lethal chondrodystrophies in man result in a common clinical syndrome including shortening of the face, mandible, and limbs. Studies of three lethal chondrodystrophic mutants in mice, viz., chondrodysplasia (cho), cartilage matrix deficiency (cmd), and disproportionate micromelia (Dmm), which share this syndrome, were performed with the aim of identifying histochemical, immunofluorescence, or ultrastructural differences which might exist among these hereditary cartilage disorders. We examined limb cartilage epiphyses from day 18 normal and mutant fetuses and observed repeatable, mostly qualitative differences. All observations were made relative to the normal control. Histochemical staining of matrix proteoglycan was moderately decreased in cho and Dmm cartilage and markedly decreased in cmd when compared to the normal control. Staining of matrix collagen was irregular in distribution in cho, increased in cmd, and decreased in Dmm. Immunofluorescence of proteoglycan was increased in the matrix of cho and Dmm and decreased in cmd. Immunofluorescence of type II collagen was heterogeneous and moderately decreased in the matrix of cho, increased in cmd, and markedly decreased in Dmm. Immunofluorescence of link protein in cho was localized in the cellular-pericellular region as in the normal and appeared increased in the matrix of cmd and Dmm. Immunofluorescence of chondronectin was localized in the cellular-pericellular region and appeared normal in all three mutants. Major differences in cellular and matrix ultrastructure were observed among the mutants, including a decreased frequency of small-diameter collagen fibrils in cho and Dmm, increased density of collagen fibrils in cmd, and dilated RER in Dmm. These observations demonstrate that distinct structural and possibly molecular differences exist among the chondrodystrophies. In the case of cmd, the differences correlated with a previously reported molecular defect, viz., absence of core protein of cartilage specific proteoglycan in the cartilage of this mutant. It is anticipated that the methods used in the present study can be applied to humans in case classification and in identifying potential mouse-human correlates.

Mucopolysaccharidosis-like cellular alterations in chondrocytes of rats treated with tilorone.

The dicationic amphiphilic compound tilorone was previously shown to interfere with the lysosomal degradation of sulfated glycosaminoglycans (GAGs) of cultured cells and to cause mucopolysaccharidosis-like alterations in several organs of rats. Since chondrocytes belong to those cells that are severely affected in cases of inherited mucopolysaccharidoses, we wished to know whether this also holds true of the experimentally induced mucopolysaccharidosis. Young rats were treated with tilorone (50-80 mg/kg of body weight) for 2-17 weeks; chondrocytes in tracheal and costal cartilage and in the epiphyseal growth plates of the tibia and ribs were examined by electron microscopy and cytochemistry (acid trimetaphosphatase and staining with Cuprolinic Blue). The tracheal and costal chondrocytes showed numerous abnormal clear vacuoles. Some of them could be identified cytochemically as lysosomes filled with polyanionic storage material, probably sulfated GAGs. In the epiphyseal plate, only the resting chondrocytes were markedly affected, whereas those of the remaining zones were hardly altered; this may be due to the short life span of these chondrocytes in rat growth plates. The present results show that chondrocytes participate in the experimental mucopolysaccharidosis and suggest that, under normal conditions, the lysosomes of chondrocytes have to cope with a considerable load of GAGs.

The ultrastructure of the organic phase associated with the inorganic substance in calcified tissues.

An organic phase is closely associated with the mineral substance is all calcified matrices, where it can be demonstrated as crystal-bound proteins by biochemical methods and as crystal ghosts by electron microscopy. Interest in crystal ghosts derives chiefly from the observation that they have the same shape, arrangement, and orientation as inorganic crystallites, which suggests they may have a role in their formation. Histochemically, crystal ghosts of epiphyseal cartilage react with colloidal iron (pH 2.0), acidic phosphotungstic acid, ruthenium red, and a number of cations including calcium, barium, magnesium, lanthanum, strontium, and terbium chloride. Their reactivity is removed by methylation and only incompletely restored by saponification. Moreover, the crystal ghosts located at the periphery of the calcified areas contain vic-glycol groups, as shown by their reactivity with periodic acid-silver nitrate and periodic acid-thiosemicarbazide-osmium. All these reactions show that the crystal ghosts of epiphyseal cartilage contain acidic, probably sulfate groups and, at least initially, vic-glycol groups. Their reactivity decreases as the calcification process is completed. Although the available data are not sufficient to allow a full understanding of the nature and function of these structures, they seem to play an important role in calcification. The hypothesis is presented that crystal ghosts are preformed in calcifying matrices and are activated by the unmasking of the reactive groups in their polymeric molecule; the unmasked groups then link up with inorganic ions in such a way to form organic-inorganic structures the inorganic ions of which are arranged in an apatitelike configuration and the filamentlike shape of which is the same as that of the polymeric molecule.